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1.  In Vitro Combination of Anti-Cytomegalovirus Compounds Acting through Different Targets: Role of the Slope Parameter and Insights into Mechanisms of Action 
Conventional therapy for human cytomegalovirus (CMV) relies on inhibition of the viral DNA polymerase. Ganciclovir (GCV) is the first-line therapy, but when GCV-resistant strains emerge, alternative therapies are extremely limited and are associated with significant toxicities. Combination of anti-CMV agents that act on different targets or stages of virus replication has not been well studied, mostly because of the limited number of anti-CMV agents. We report our investigation of combinations of agents that inhibit CMV by targeting the viral DNA polymerase, cellular kinases, or other cell/virus mechanisms yet to be discovered. The selected compounds differed by the slopes of their dose-response curve: compounds with a slope of 1 (GCV) representing one target or noncooperativity and compounds with high slopes indicating positive cooperativity. Analysis of anti-CMV drug combinations using the Bliss model (which accounts for the slope parameter) distinguished between combinations with synergistic, antagonistic, and additive activities. The combination of GCV and foscarnet was slightly synergistic; strong synergism was found when GCV was used with artemisinin-derived monomers or dimers or the MEK inhibitor U0126. The combination of GCV and cardiac glycosides (digoxin, digitoxin, and ouabain) was additive. The monomeric artemisinin artesunate was synergistic when combined with U0126 or the multikinase inhibitor sunitinib. However, the combination of artemisinin-derived dimers (molecular weights, 606 and 838) and U0126 or sunitinib was antagonistic. These results demonstrate that members of a specific drug class show similar patterns of combination with GCV and that the slope parameter plays an important role in the evaluation of drug combinations. Lastly, antagonism between different classes of CMV inhibitors may assist in target identification and improve the understanding of CMV inhibition by novel compounds.
doi:10.1128/AAC.01972-13
PMCID: PMC3910867  PMID: 24277030
2.  Activation of Nucleotide Oligomerization Domain 2 (NOD2) by Human Cytomegalovirus Initiates Innate Immune Responses and Restricts Virus Replication 
PLoS ONE  2014;9(3):e92704.
Nucleotide-binding oligomerization domain 2 (NOD2) is an important innate immune sensor of bacterial pathogens. Its induction results in activation of the classic NF-κB pathway and alternative pathways including type I IFN and autophagy. Although the importance of NOD2 in recognizing RNA viruses has recently been identified, its role in sensing DNA viruses has not been studied. We report that infection with human cytomegalovirus (HCMV) results in significant induction of NOD2 expression, beginning as early as 2 hours post infection and increasing steadily 24 hours post infection and afterwards. Infection with human herpesvirus 1 and 2 does not induce NOD2 expression. While the HCMV-encoded glycoprotein B is not required for NOD2 induction, a replication competent virion is necessary. Lentivirus-based NOD2 knockdown in human foreskin fibroblasts (HFFs) and U373 glioma cells leads to enhanced HCMV replication along with decreased levels of interferon beta (IFN-β) and the pro-inflammatory cytokine, IL8. NOD2 induction in HCMV-infected cells activates downstream NF-κB and interferon pathways supported by reduced nuclear localization of NF-κB and pIRF3 in NOD2 knockdown HFFs. Stable overexpression of NOD2 in HFFs restricts HCMV replication in association with increased levels of IFN-β and IL8. Similarly, transient overexpression of NOD2 in U373 cells or its downstream kinase, RIPK2, results in decreased HCMV replication and enhanced cytokine responses. However, overexpression of a mutant NOD2, 3020insC, associated with severe Crohn's disease, results in enhanced HCMV replication and decreased levels of IFN-β in U373 cells. These results show for the first time that NOD2 plays a significant role in HCMV replication and may provide a model for studies of HCMV recognition by the host cell and HCMV colitis in Crohn's disease.
doi:10.1371/journal.pone.0092704
PMCID: PMC3966837  PMID: 24671169
3.  Unique and Highly Selective Anticytomegalovirus Activities of Artemisinin-Derived Dimer Diphenyl Phosphate Stem from Combination of Dimer Unit and a Diphenyl Phosphate Moiety 
We report that the artemisinin-derived dimer diphenyl phosphate (DPP; dimer 838) is the most selective inhibitor of human cytomegalovirus (CMV) replication among a series of artemisinin-derived monomers and dimers. Dimer 838 was also unique in being an irreversible CMV inhibitor. The peroxide unit within artemisinins' chemical structures is critical to their activities, and its absence results in loss of anti-CMV activities. Surprisingly, the deoxy dimer of 838 retained modest anti-CMV activity, suggesting that the DPP moiety of dimer 838 contributes to its anti-CMV activities. DPP alone did not inhibit CMV replication, but triphenyl phosphate (TPP) had modest CMV inhibition, although its selectivity index was low. Artemisinin DPP derivatives dimer 838 and monomer diphenyl phosphate (compound 558) showed stronger CMV inhibition and a higher selectivity index than their analogs lacking the DPP unit. An add-on and removal assay revealed that removing DPP derivatives (compounds 558 and 838) but not the non-DPP backbones (artesunate and compound 606) at 24 h postinfection (hpi) already resulted in dominant CMV inhibition. CMV inhibition was fully irreversible with 838 and partially irreversible with 558, while non-DPP artemisinins were reversible inhibitors. While all artemisinin derivatives and TPP reduced the expression of the CMV immediate early 2 (IE2), UL44, and pp65 proteins at or after 48 hpi, only TPP inhibited the expression of both IE1 and IE2. Combination of a non-DPP dimer (compound 606) with TPP was synergistic in CMV inhibition, while ganciclovir and TPP were additive. Although TPP shared structural similarity with monomer DPP (compound 558) and dimer DPP (compound 838), its pattern of CMV inhibition was significantly different from the patterns of the artemisinins. These findings demonstrate that the DPP group contributes to the unique activities of compound 838.
doi:10.1128/AAC.00893-13
PMCID: PMC3754318  PMID: 23774439
4.  Detecting Respiratory Viruses in Asymptomatic Children 
BACKGROUND
Viral respiratory infections are among the most common reasons for hospitalization of children in the United States. Our objective was to compare molecular and conventional methods in a cohort of hospitalized children with and without symptoms of respiratory viral illness (RVI).
METHODS
Retrospective cohort study of infants and toddlers hospitalized between December 2007 and March 2008 at Johns Hopkins Hospital. 569 of 641 patient visits (89%) were tested on admission. Conventional tests (immunochromatography, direct fluorescent antibody, shell vial, and tube culture) were performed on all patients and nucleic acid tests (NATs) were performed on available samples (n=306). Viruses were grouped into those routinely (Group 1) and those not routinely (Group 2) detected by conventional methods.
RESULTS
In children with RVI symptoms (N=148), NATs identified a virus in 83% specimens compared with 49% by conventional methods (p<0.001), but detected a similar percentage of specimens with Group 1 viruses (48.6% and 55.4%, p=0.13) compared with conventional tests. In children without RVI symptoms (N=158), NATs identified a virus in 41.7% specimens compared with 4.4% by conventional tests (p<0.001) and identified more Group 1 viruses (9.5% and 4.4%, p=0.03) compared with conventional tests. Group 2 viruses were identified by NATs in a similar percentage of symptomatic and asymptomatic patients (25% and 32.3%, p=0.20).
CONCLUSION
Molecular assays may have several advantages over conventional methods for detecting respiratory viruses, including improved sensitivity and rapid detection, but given the high prevalence of positive results in children without RVI symptoms, results should be interpreted cautiously.
doi:10.1097/INF.0b013e318265a804
PMCID: PMC3505556  PMID: 22739572
molecular diagnostic methods; pediatric respiratory infections; nucleic acid tests; virus isolation
5.  Wnt Modulating Agents Inhibit Human Cytomegalovirus Replication 
Infection with human cytomegalovirus (HCMV) continues to be a threat for pregnant women and immunocompromised hosts. Although limited anti-HCMV therapies are available, development of new agents is desired. The Wnt signaling pathway plays a critical role in embryonic and cancer stem cell development and is targeted by gammaherpesviruses, Epstein-Barr virus (EBV), and Kaposi's sarcoma-associated herpesvirus (KSHV). HCMV infects stem cells, including neural progenitor cells, during embryogenesis. To investigate the role of Wnt in HCMV replication in vitro, we tested monensin, nigericin, and salinomycin, compounds that inhibit cancer stem cell growth by modulating the Wnt pathway. These compounds inhibited the replication of HCMV Towne and a clinical isolate. Inhibition occurred prior to DNA replication but persisted throughout the full replication cycle. There was a significant decrease in expression of IE2, UL44, and pp65 proteins. HCMV infection resulted in a significant and sustained decrease in expression of phosphorylated and total lipoprotein receptor-related protein 6 (pLRP6 and LRP6, respectively), Wnt 5a/b, and β-catenin and a modest decrease in Dvl2/3, while levels of the negative regulator axin 1 were increased. Nigericin decreased the expression of pLRP6, LRP6, axin 1, and Wnt 5a/b in noninfected and HCMV-infected cells. For all three compounds, a correlation was found between expression levels of Wnt 5a/b and axin 1 and HCMV inhibition. The decrease in Wnt 5a/b and axin 1 expression was more significant in HCMV-infected cells than noninfected cells. These data illustrate the complex effects of HCMV on the Wnt pathway and the fine balance between Wnt and HCMV, resulting in abrogation of HCMV replication. Additional studies are required to elucidate how HCMV targets Wnt for its benefit.
doi:10.1128/AAC.00029-13
PMCID: PMC3716142  PMID: 23571549
6.  Dried Urine Spots for Detection and Quantification of Cytomegalovirus in Newborns 
Dried urine spots (DUS) have been reported to provide a simple screening tool for congenital cytomegalovirus (CMV) infection. We developed a standardized method for CMV quantification from DUS. Two applications of 20 μ L urine remained within the rim of the filter paper disc and were used to determine the analytical performance of Towne CMV spiked into urine and applied on the discs. The measurable range spanned 3.7 to ≥8.0 log10 copies/mL. The detection limit was 22 DNA copies/disc. Urine samples from congenitally-infected newborns and negative controls were either diluted 1:10 or applied on filter paper at the same volume. DNA copy number from DUS correlated well with copy number from 1:10 diluted urine, although there was a trend for lower levels from DUS (0.3 log10 difference). Our standardized method for CMV detection and quantification may facilitate CMV studies in resource-limited areas and allow for longitudinal monitoring of viral loads in treated infants.
doi:10.1016/j.diagmicrobio.2012.04.009
PMCID: PMC3396794  PMID: 22658885
7.  Clinical Accuracy of a PLEX-ID Flu Device for Simultaneous Detection and Identification of Influenza Viruses A and B 
Respiratory tract infections caused by influenza A and B viruses often present nonspecifically, and a rapid, high-throughput laboratory technique that can identify influenza viruses is clinically and epidemiologically desirable. The PLEX-ID Flu assay (Abbott Molecular Inc., Des Plaines, IL) incorporates multilocus PCR and electrospray ionization-mass spectrometry to detect and differentiate influenza A 2009 H1N1 (H1N1-p), seasonal H1N1 (H1N1-s), influenza A H3N2, and influenza B viruses in nasopharyngeal swab (NPS) specimens. The clinical performance characteristics of the PLEX-ID Flu assay in symptomatic patients were determined in this multicenter trial. A total of 2,617 prospectively and retrospectively collected NPS specimens from patients with influenza-like illness between February 2008 and 28 May 2010 were eligible for inclusion in the study. Each specimen was tested in parallel by the PLEX-ID Flu assay and by the Prodesse ProFLU+ assay (Prodesse Inc., Madison, WI), to detect influenza A and B viruses. Specimens testing positive for influenza A virus by ProFLU+ were subtyped as H1N1-p, H1N1-s, or H3N2 by using the ProFAST+ assay (Gen-Probe Prodesse Inc.). The reproducibility of the PLEX-ID Flu assay ranged from 98.3 to 100.0%, as determined by testing a nine-specimen panel at three clinical sites on each of 5 days. Positive percent agreements (PPAs) and negative percent agreements (NPAs) of the PLEX-ID Flu assay were 94.5% and 99.0% for influenza A virus and 96.0% and 99.9% for influenza B virus, respectively. For the influenza A virus subtyping characterization, the PLEX-ID Flu assay had PPAs and NPAs of 98.3% and 97.5% for H1N1-p, 88.6% and 100.0% for H1N1-s, and 98.0% and 99.9% for H3N2, respectively. The overall agreements between the PLEX-ID and Prodesse ProFLU+/ProFAST+ assays were 97.1 to 100.0%. Bidirectional Sanger sequencing analysis revealed that 87.5% of 96 discrepant results between the PLEX-ID Flu and ProFLU+/ProFAST+ assays were found upon influenza A virus detection and H1N1-p subtyping. The PLEX-ID Flu assay demonstrated a high level of accuracy for the simultaneous detection and identification of influenza A and B viruses in patient specimens, providing a new laboratory tool for the rapid diagnosis and management of influenza A and B virus infections.
doi:10.1128/JCM.01978-12
PMCID: PMC3536229  PMID: 23077123
8.  Diagnostic performance of two highly multiplexed respiratory virus assays in a pediatric cohort 
Background
Rapid detection of respiratory viruses is important for management and infection control in hospitalized patients. Multiplex nucleic acid tests (NATs) have begun to replace conventional methods as gold standards for respiratory virus detection.
Objective
To compare the performance of two large multiplex NATS, ResPlex II (RPII) and Respiratory Virus Surveillance kit with electrospray ionization mass spectrometry (RVS/MS) using nasopharyngeal aspirates (NPAs) from hospitalized children who had been tested previously with conventional methods.
Study design
Stored residual NPAs (N = 306) were tested concomitantly by RPII and RVS/MS. Alternate NATs were used to adjudicate discordant results.
Results
More viruses were detected with multiplex NATs (RPII, 110; RVS/MS, 109) than conventional assays (86); diagnostic gain was primarily for fastidious viruses (coronaviruses and enteroviruses [EVs]/human rhinoviruses [HRVs]). Total positive and negative agreement between the multiplex NATs for all viruses detected was quite high (86% positive agreement, 99% negative agreement). Most individual viruses were detected with fairly equivalent accuracy by the multiplex NATs, except for adenoviruses (RPII sensitivity 40%) and human metapneumovirus (RVS/MS sensitivity 42%). RPII had the advantage of detecting EVs and HRVs, however, it demonstrated considerable EV/HRV cross-reactivity (29 HRV-positive specimens by real-time PCR were positive for EV by RPII and 21 specimens positive for HRV only by RT-PCR were dual positive for EV/HRV by RPII). RPII also had reduced sensitivity for HRV detection (in 36 specimens, HRV was detected by RT-PCR but not by RPII).
Conclusions
Both multiplex NATs were promising, but had notable limitations.
doi:10.1016/j.jcv.2012.06.019
PMCID: PMC3586269  PMID: 22832060
Respiratory virus diagnostics; Respiratory virus nucleic acid tests; Respiratory virus multiplex tests
9.  Human Cytomegalovirus Inhibition by Cardiac Glycosides: Evidence for Involvement of the hERG Gene 
Infection with human cytomegalovirus (HCMV) continues to be a major threat for pregnant women and the immunocompromised population. Although several anti-HCMV therapies are available, the development of new anti-HCMV agents is highly desired. There is growing interest in identifying compounds that might inhibit HCMV by modulating the cellular milieu. Interest in cardiac glycosides (CG), used in patients with congestive heart failure, has increased because of their established anticancer and their suggested antiviral activities. We report that the several CG—digoxin, digitoxin, and ouabain—are potent inhibitors of HCMV at nM concentrations. HCMV inhibition occurred prior to DNA replication, but following binding to its cellular receptors. The levels of immediate early, early, and late viral proteins and cellular NF-κB were significantly reduced in CG-treated cells. The activity of CG in infected cells correlated with the expression of the potassium channel gene, hERG. CMV infection upregulated hERG, whereas CG significantly downregulated its expression. Infection with mouse CMV upregulated mouse ERG (mERG), but treatment with CG did not inhibit virus replication or mERG transcription. These findings suggest that CG may inhibit HCMV by modulating human cellular targets associated with hERG and that these compounds should be studied for their antiviral activities.
doi:10.1128/AAC.00898-12
PMCID: PMC3421849  PMID: 22777050
10.  Artemisinin-Derived Dimer Diphenyl Phosphate Is an Irreversible Inhibitor of Human Cytomegalovirus Replication 
We previously reported that among a series of artemisinin-derived monomers and dimers, dimer diphenyl phosphate (838) was the most potent inhibitor of human cytomegalovirus (CMV) replication. Our continued investigation of a prototypic artemisinin monomer (artesunate [AS]) and dimer (838) now reveals that both compounds have specific activity against CMV but do not inhibit lytic replication of human herpesvirus 1 or 2 or Epstein-Barr virus. AS and 838 inhibited CMV replication during the first 24 h of the virus replication cycle, earlier than the time of ganciclovir (GCV) activities and prior to DNA synthesis. Neither compound inhibited virus entry. Quantification of DNA replication and virus yield revealed a similar level of inhibition by GCV, but AS and 838 had a 10-fold-higher inhibition of virus yield than of DNA replication, suggesting that artemisinins could inhibit CMV through multiple steps: a predominant early inhibition and possibly an additional step following DNA replication. During the strong early CMV inhibition, the transcription of immediate-early genes was not significantly downregulated, and viral protein expression was reduced only after 48 h. AS and GCV were reversible CMV inhibitors, but the inhibition of CMV replication by 838 was irreversible. Combinations of GCV and 838 as well as GCV and AS were highly synergistic. Finally, treatment with 838, but not AS, prior to CMV infection demonstrated strong anti-CMV activity. These findings illustrate the unique activities of dimer 838, including early and irreversible CMV inhibition, possibly by tight binding to its target.
doi:10.1128/AAC.00519-12
PMCID: PMC3393438  PMID: 22547612
11.  Cytomegalovirus DNA Quantification Using an Automated Platform for Nucleic Acid Extraction and Real-Time PCR Assay Setup ▿ 
Journal of Clinical Microbiology  2011;49(7):2703-2705.
Analytical performance characteristics of the QIAsymphony RGQ system with artus cytomegalovirus (CMV) reagents were determined. Measurable range spanned 2.0 to ≥7.0 log10 copies/ml. The detection limit was 23 copies/ml. Intrarun and interrun coefficients of variation were ≤2.1% at 3.0 and 5.0 log10 copies/ml. In clinical specimens, RGQ values were ∼0.2 log10 copies/ml higher than those in an assay using a BioRobot M48 extraction/manual reaction setup/7500 Real-Time PCR instrument. No cross-contamination was observed.
doi:10.1128/JCM.00721-11
PMCID: PMC3147859  PMID: 21562101
12.  Recombinant luciferase-expressing human cytomegalovirus (CMV) for evaluation of CMV inhibitors 
Virology Journal  2011;8:40.
Recombinant Towne CMV expressing luciferase under the control of CMV-DNA polymerase (POL) or the late pp28 (UL99) promoters were evaluated for potential application in high-throughput screening of anti-viral compounds. POL-and pp28-luciferase displayed maximal expression 48 and 72 hours post infection, respectively. The pp28-luciferase virus achieved a wider dynamic range of luciferase expression (6-7 logs) and was selected for testing of inhibition by five anti-viral compounds. Luciferase expression highly correlated with plaque reduction and real-time PCR. The pp28-luciferase reporter system is rapid, reproducible, and highly sensitive. It may be applied to screening of novel anti-CMV compounds.
doi:10.1186/1743-422X-8-40
PMCID: PMC3041771  PMID: 21269468
13.  Detection of a Single Identical Cytomegalovirus (CMV) Strain in Recently Seroconverted Young Women 
PLoS ONE  2011;6(1):e15949.
Background
Infection with multiple CMV strains is common in immunocompromised hosts, but its occurrence in normal hosts has not been well-studied.
Methods
We analyzed CMV strains longitudinally in women who acquired CMV while enrolled in a CMV glycoprotein B (gB) vaccine trial. Sequencing of four variable genes was performed in samples collected from seroconversion and up to 34 months thereafter.
Results
199 cultured isolates from 53 women and 65 original fluids from a subset of 19 women were sequenced. 51 women were infected with one strain each without evidence for genetic drift; only two women shed multiple strains. Genetic variability among strains increased with the number of sequenced genetic loci. Nevertheless, 13 of 53 women proved to be infected with an identical CMV strain based on sequencing at all four variable genes. CMV vaccine did not alter the degree of genetic diversity amongst strains.
Conclusions
Primary CMV infection in healthy women nearly always involves shedding of one strain that remains stable over time. Immunization with CMVgB-1 vaccine strain is not selective against specific strains. Although 75% of women harbored their unique strain, or a strain shared with only one other woman, 25% shared a single common strain, suggesting that this predominant strain with a particular combination of genetic loci is advantageous in this large urban area.
doi:10.1371/journal.pone.0015949
PMCID: PMC3018470  PMID: 21264339
14.  Mortality associated with resistant cytomegalovirus among patients with cytomegalovirus retinitis and AIDS 
Ophthalmology  2009;117(1):128.
Objective
To evaluate the effect of drug-resistant cytomegalovirus (CMV) on survival among patients with CMV retinitis.
Design
Prospective cohort study during 1993–2003.
Participants
266 patients with AIDS and newly diagnosed CMV retinitis treated with either ganciclovir or foscarnet.
Methods
Data on ganciclovir and foscarnet resistance were obtained from blood and urine specimens collected at regular, pre-determined intervals. The effect of resistant CMV on mortality was evaluated with a time-dependent Cox proportional hazard model.
Main outcome measure
Mortality
Results
Median survival of the entire cohort was 12.6 months. Analysis of risk factors for mortality demonstrated that resistant CMV was associated with an increased mortality (hazard ratio = 1.65, 95% confidence interval=1.05–2.56, P=0.032). Among the other parameters tested, only time since AIDS diagnosis was associated significantly with mortality, with a hazard ratio of 1.10 per year since AIDS diagnosis (P=0.001).
Conclusions
Resistant CMV is associated with increased mortality among patients with AIDS being treated for CMV retinitis.
doi:10.1016/j.ophtha.2009.06.016
PMCID: PMC2815221  PMID: 19818505
AIDS; cytomegalovirus; retinitis; resistance; mortality
15.  Gangliosides Block Aggregatibacter Actinomycetemcomitans Leukotoxin (LtxA)-Mediated Hemolysis  
Toxins  2010;2(12):2824-2836.
Aggregatibacter actinomycetemcomitans is an oral pathogen and etiologic agent of localized aggressive periodontitis. The bacterium is also a cardiovascular pathogen causing infective endocarditis. A. actinomycetemcomitans produces leukotoxin (LtxA), an important virulence factor that targets white blood cells (WBCs) and plays a role in immune evasion during disease. The functional receptor for LtxA on WBCs is leukocyte function antigen-1 (LFA-1), a β-2 integrin that is modified with N-linked carbohydrates. Interaction between toxin and receptor leads to cell death. We recently discovered that LtxA can also lyse red blood cells (RBCs) and hemolysis may be important for pathogenesis of A. actinomycetemcomitans. In this study, we further investigated how LtxA might recognize and lyse RBCs. We found that, in contrast to a related toxin, E. coli α-hemolysin, LtxA does not recognize glycophorin on RBCs. However, gangliosides were able to completely block LtxA-mediated hemolysis. Furthermore, LtxA did not show a preference for any individual ganglioside. LtxA also bound to ganglioside-rich C6 rat glioma cells, but did not kill them. Interaction between LtxA and C6 cells could be blocked by gangliosides with no apparent specificity. Gangliosides were only partially effective at preventing LtxA-mediated cytotoxicity of WBCs, and the effect was only observed when a high ratio of ganglioside:LtxA was used over a short incubation period. Based on the results presented here, we suggest that because of the similarity between N-linked sugars on LFA-1 and the structures of gangliosides, LtxA may have acquired the ability to lyse RBCs.
doi:10.3390/toxins2122824
PMCID: PMC3153184  PMID: 22069577
erythrocytes; toxin; periodontal disease; endocarditis; RTX toxin
16.  Artemisinin-Derived Dimers Have Greatly Improved Anti-Cytomegalovirus Activity Compared to Artemisinin Monomers 
PLoS ONE  2010;5(4):e10370.
Background
Artesunate, an artemisinin-derived monomer, was reported to inhibit Cytomegalovirus (CMV) replication. We aimed to compare the in-vitro anti-CMV activity of several artemisinin-derived monomers and newly synthesized artemisinin dimers.
Methods
Four artemisinin monomers and two novel artemisinin-derived dimers were tested for anti-CMV activity in human fibroblasts infected with luciferase-tagged highly–passaged laboratory adapted strain (Towne), and a clinical CMV isolate. Compounds were evaluated for CMV inhibition and cytotoxicity.
Results
Artemisinin dimers effectively inhibited CMV replication in human foreskin fibroblasts and human embryonic lung fibroblasts (EC50 for dimer sulfone carbamate and dimer primary alcohol 0.06±0.00 µM and 0.15±0.02 µM respectively, in human foreskin fibroblasts) with no cytotxicity at concentrations required for complete CMV inhibition. All four artemisinin monomers (artemisinin, artesunate, artemether and artefanilide) shared a similar degree of CMV inhibition amongst themselves (in µM concentrations) which was significantly less than the inhibition achieved with artemisinin dimers (P<0.0001). Similar to monomers, inhibition of CMV with artemisinin dimers appeared early in the virus life cycle as reflected by decreased expression of the immediate early (IE1) protein.
Conclusions
Artemisinin dimers are potent and non-cytotoxic inhibitors of CMV replication. These compounds should be studied as potential therapeutic agents for the treatment of CMV infection in humans.
doi:10.1371/journal.pone.0010370
PMCID: PMC2860993  PMID: 20442781
17.  Differential CMV-Specific CD8+ Effector T Cell Responses in the Lung Allograft Predominate over the Blood during Human Primary Infection1 
Acquisition of T cell responses during primary CMV infection in lung transplant recipients (LTRs) appear critical for host defense and allograft durability, with increased mortality in donor+/recipient− (D+R−) individuals. In 15 D+R− LTRs studied, acute primary CMV infection was characterized by viremia in the presence or absence of pneumonitis, with viral loads higher in the lung airways/allograft compared with the blood. A striking influx of CD8+ T cells into the lung airways/allograft was observed, with inversion of the CD4+:CD8+ T cell ratio. De novo CMV-specific CD8+ effector frequencies in response to pooled peptides of pp65 were strikingly higher in lung mononuclear cells compared with the PBMC and predominated over IE1-specific responses and CD4+ effector responses in both compartments. The frequencies of pp65-specific cytokine responses were significantly higher in lung mononuclear cells compared with PBMC and demonstrated marked contraction with long-term persistence of effector memory CD8+ T cells in the lung airways following primary infection. CMV-tetramer+CD8+ T cells from PBMC were CD45RA− during viremia and transitioned to CD45RA+ following resolution. In contrast, CMV-specific CD8+ effectors in the lung airways/allograft maintained a CD45RA− phenotype during transition from acute into chronic infection. Together, these data reveal differential CMV-specific CD8+ effector frequencies, immunodominance, and polyfunctional cytokine responses predominating in the lung airways/allograft compared with the blood during acute primary infection. Moreover, we show intercompartmental phenotypic differences in CMV-specific memory responses during the transition to chronic infection.
PMCID: PMC2668691  PMID: 18566421
18.  Verification of an Assay for Quantification of Hepatitis C Virus RNA by Use of an Analyte-Specific Reagent and Two Different Extraction Methods 
Journal of Clinical Microbiology  2004;42(8):3581-3588.
Protocols were designed for quantification and detection of hepatitis C virus (HCV) RNA by the use of an analyte-specific reagent (ASR) (Roche COBAS TaqMan48 [CTM48] HCV) after manual and automated RNA extraction. The purposes were to determine (i) assay performance characteristics using manual and automated RNA extraction methods, (ii) whether measurable range and limit of detection (LOD) of the ASR assay were influenced by genotype, and (iii) correlation of quantification by CTM48 HCV ASR and COBAS Monitor HCV v. 2.0. For HCV genotype 1 (Gt1), the lower limits of quantification after manual extraction were slightly lower than those for automated extraction (1.0 versus 1.5 log10 IU/ml). Results were linear up to the highest concentration tested after extraction by both methods (manual, 6.1 log10; automated, 6.4 log10). Similar results were obtained for Gt2 (1.8 to 6.8 log10 IU/ml) and Gt3 (1.6 to 6.8 log10 IU/ml) after automated extraction. The LOD of Gt1 virus was 10 IU/ml after manual extraction and between 25 and 37.5 IU/ml after automated extraction. Results with Gt2 and Gt3 viruses were similar after automated extraction (Gt2, between 25 and 50 IU/ml; Gt3, 25 IU/ml). Variability (intrarun and interrun, at concentrations throughout the range of quantification) was ≤13% for both extraction methods. Clinical specimens tested by Monitor were quantified using the CTM48 HCV ASR assay. Characteristics of the regression line included a slope of 0.98 and y intercept of −0.23. Quantification by the two methods was correlated (r = 0.97). CTM48 HCV ASR assay values were on average twofold lower than those for Monitor HCV v. 2.0. These data suggest that our assay combines the characteristics of qualitative and quantitative PCR platforms into a single test.
doi:10.1128/JCM.42.8.3581-3588.2004
PMCID: PMC497620  PMID: 15297501
19.  Comparison of Automated and Manual Nucleic Acid Extraction Methods for Detection of Enterovirus RNA 
Journal of Clinical Microbiology  2003;41(8):3532-3536.
Automated nucleic acid extraction is an attractive alternative to labor-intensive manual methods. We compared two automated methods, the BioRobot M48 instrument (Qiagen, Inc.) and MagNA Pure (Roche Applied Sciences) methods, to two manual methods, the QIAamp Viral RNA Mini kit (Qiagen) and TRIzol (Invitrogen), for the extraction of enterovirus RNA. Analytical sensitivity was assessed by dilution analysis of poliovirus type 2 Sabin in cerebrospinal fluid. The sensitivity of PCR was equivalent after RNA extraction with QIAamp, BioRobot M48, and MagNA Pure. All 18 replicates of 100 PFU/ml were detected after extraction by the four methods. Fewer replicates of each successive dilution were detected after extraction by each method. At 10−1 PFU/ml, 17 of 18 replicates were positive by QIAamp, 15 of 18 replicates were positive by BioRobot M48, and 12 of 18 replicates were positive by MagNA Pure; at 10−2 PFU/ml, 4 of 17 replicates were positive by QIAamp, 2 of 18 replicates were positive by BioRobot M48, and 0 of 18 replicates were positive by MagNA Pure. At 10−3 PFU/ml, no replicates were detected. Evaluation of TRIzol was discontinued after nine replicates due to a trend of lower sensitivity (at 10−3 PFU/ml eight of nine replicates were positive at 100 PFU/ml, four of nine replicates were positive at 10−1 PFU/ml, and zero of nine replicates were positive at 10−2 PFU/ml). Concordant results were obtained in 24 of 28 clinical specimens after extraction by all methods. No evidence of contamination was observed after extraction by automated instruments. The data indicate that the sensitivity of enterovirus PCR is largely similar after extraction by QIAamp, BioRobot M48, and MagNA Pure; a trend of decreased sensitivity was observed after TRIzol extraction. However, the results of enterovirus PCR were largely concordant in patient samples, indicating that the four extraction methods are suitable for detection of enteroviruses in clinical specimens.
doi:10.1128/JCM.41.8.3532-3536.2003
PMCID: PMC179781  PMID: 12904351
20.  Confirmation of Low-Titer, Herpes Simplex Virus-Positive Specimen Results by the Enzyme-Linked Virus-Inducible System (ELVIS) Using PCR and Repeat Testing 
Journal of Clinical Microbiology  1999;37(12):3986-3989.
The ELVIS HSV Id test kit (an enzyme-linked virus-inducible system) (Diagnostic Hybrids, Inc.) uses genetically engineered BHK cells to produce a detectable enzyme, beta-galactosidase, upon infection with either herpes simplex virus (HSV) type 1 (HSV-1) or HSV-2. Twenty six ELVIS-positive clinical specimens were selected for study by PCR and with monoclonal antibodies because they were originally low-titer HSV-positive specimens by ELVIS but HSV antibody nonreactive upon follow-up staining of the ELVIS monolayer. Twenty-one of 26 specimens were frozen, thawed, and retested with ELVIS without removing the cellular debris from the specimen; 18 were ELVIS positive and 3 were ELVIS negative on retesting. A typing result was provided upon retesting for 14 of 18 ELVIS-positive specimens (11 were HSV-1 and 3 were HSV-2) with HSV-specific monoclonal antibodies; no antibody signal was observed for 4 of 18 ELVIS-positive specimens. Sixteen of 26 specimens were subjected to blinded PCR analysis with two different primer sets, including all those that were repeat tested with ELVIS without success and those that had insufficient quantity for repeat testing. All 16 specimens analyzed were PCR positive with primer set 1; 15 of 16 were also positive with primer set 2, with the HSV type identified for all specimens (7 were HSV-1 and 8 were HSV-2). These results indicate that the original ELVIS result with these low-titer specimens was correct and further confirm the sensitivity and specificity of ELVIS HSV Id as a rapid, cell culture-based kit for the detection of HSV.
PMCID: PMC85862  PMID: 10565919
21.  Comparison of Cytomegalovirus Loads in Plasma and Leukocytes of Patients with Cytomegalovirus Retinitis 
Journal of Clinical Microbiology  1999;37(5):1431-1435.
Cytomegalovirus (CMV) DNA loads in paired leukocyte and plasma samples from 199 patient visits by 66 patients with CMV retinitis were determined. Leukocyte CMV load determinations had a greater range of values (mean, 24,587 copies/106 leukocytes; maximum, 539,000) than did plasma CMV load determinations (mean, 10,302 copies/ml; maximum, 386,000), and leukocyte viral loads were detectable in a greater proportion of patients at the time of diagnosis of CMV retinitis prior to initiation of anti-CMV therapy (82%) than were plasma viral loads (64%) (P = 0.0078). Agreement with CMV blood cultures was slightly better for plasma (κ = 0.68) than for leukocytes (κ = 0.53), due to a greater proportion of patients with detectable viral loads in leukocytes having negative blood cultures.
PMCID: PMC84793  PMID: 10203500
22.  PCR Method for Detection of Adenovirus in Urine of Healthy and Human Immunodeficiency Virus-Infected Individuals 
Journal of Clinical Microbiology  1998;36(11):3323-3326.
Adenoviruses (AdV) cause diseases that range from localized, self-limited illnesses to fatal infections in immunocompromised patients. Culture is assumed to be sensitive but requires viable virus and up to 3 weeks for detection, and it can be inhibited by bacterial contamination. A new PCR method amplifying a region of the hexon gene was developed in order to detect AdV in urine more rapidly and with greater sensitivity than obtainable by culture technology. All 18 serotypes tested were detected. Quantitatively, with optimized urine processing, AdV PCR detected 0.2 PFU/ml (serotype 11) and 10 DNA copies/ml (serotype 2). Serially collected urine samples from human immunodeficiency virus (HIV)-infected patients with concurrent cytomegalovirus retinitis were divided into three groups: AdV culture-positive samples, AdV culture-negative or bacterially contaminated samples from patients with a history of AdV culture-positive urines, and AdV culture-negative samples from patients without a history of AdV culture positivity. Urine samples from healthy adults were also tested by culture and PCR to screen for asymptomatic shedding. Amplification was assessed with and without prior DNA purification. AdV was detected by PCR in 90% of culture-positive urines (100% of unclotted samples, e.g., those culture positive after storage for PCR testing), 71% of culture-negative or bacterially contaminated urines from AdV-infected patients, and 28% from AdV culture-negative patients. Healthy volunteers were culture negative for AdV, and 96% were PCR negative. The new AdV PCR method is rapid and sensitive and can detect viral DNA in samples for which culturing is problematic. The role of AdV replication during HIV infection merits further investigation with sensitive tools such as PCR.
PMCID: PMC105322  PMID: 9774586
23.  Incidence of Foscarnet Resistance and Cidofovir Resistance in Patients Treated for Cytomegalovirus Retinitis 
Cytomegalovirus (CMV) retinitis is a common opportunistic infection in patients with AIDS. With long-term therapy for CMV retinitis, resistant CMV may develop. In a prospective study of 122 patients with CMV retinitis, 2.4 and 0.8% of patients had foscarnet-resistant blood culture isolates (50% inhibitory concentration [IC50], >400 μM) and urine culture isolates, respectively, at diagnosis of CMV retinitis prior to treatment, whereas 4.1 and 6.6% had cidofovir-resistant (IC50, >2 μM) blood and urine culture isolates, respectively. Patients were treated according to best medical judgement. Of 44 foscarnet-treated patients, 26% had a resistant blood or urine culture isolate by 6 months of treatment and 37% had a resistant isolate by 9 months; of 13 cidofovir-treated patients, 29% had a resistant blood or urine culture isolate by 3 months of therapy. The probabilities of developing foscarnet resistance while on foscarnet and developing cidofovir resistance while on cidofovir were not significantly different from that for developing ganciclovir resistance while on ganciclovir (odds ratios, 1.87 [P = 0.19] and 2.28 [P = 0.15], respectively).
PMCID: PMC105794  PMID: 9736542

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